Microbiol Monogr (9)J. Tachezy: Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic EukaryotesDOI 10.1007/7171_2007_104/Published online: 5 December 2007© Springer-Verlag Berlin Heidelberg 2007

Structure of the Hydrogenosome

Marlene Benchimol

Universidade Santa Úrsula, Rua Jornalista Orlando Dantas 59,CEP 222-31-010 Rio de Janeiro, Brazilmarleneben@uol.com.br

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

2 Where are Hydrogenosomes Found? . . . . . . . . . . . . . . . . . . . . . . 76

3 Hydrogenosome Shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

4 Hydrogenosome Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

5 Hydrogenosome Components . . . . . . . . . . . . . . . . . . . . . . . . . 795.1 The Hydrogenosome Envelope . . . . . . . . . . . . . . . . . . . . . . . . . 795.2 The Peripheral Vesicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815.3 The Matrix of the Hydrogenosome . . . . . . . . . . . . . . . . . . . . . . . 84

6 Fungal Hydrogenosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

7 Proximity with Other Cellular Structures . . . . . . . . . . . . . . . . . . . 877.1 Hydrogenosomes and Endoplasmic Reticulum . . . . . . . . . . . . . . . . 87

8 Hydrogenosome Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . 88

9 Hydrogenosome Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

10 Hydrogenosome Behavior in the Cell Cycle . . . . . . . . . . . . . . . . . . 92

11 Hydrogenosomes Connection to Microtubules . . . . . . . . . . . . . . . . 93

12 Immunolabeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Abstract Hydrogenosomes are very interesting organelles found in nonmitochondrial or-ganisms. They display similarities and differences with mitochondria. Hydrogenosomesare spherical or slightly elongated organelles, although very elongated hydrogenosomesare also found. They measure between 200 and 1000 nm, but under stress conditions canreach 2 µm. Hydrogenosomes divide in three different ways, like mitochondria: segmen-tation, partition, and the heart form. They may divide at any phase of the cell cycle.Nucleoid or electron-dense deposits are not considered part of the normal structureof the hydrogenosome. Hydrogenosomes are surrounded by two closely apposed mem-branes and present a granular matrix. Hydrogenosomes have one or multiple peripheralvesicles, which incorporate calcium. The peripheral vesicle can be isolated from thehydrogenosomal matrix and is considered a distinct hydrogenosomal compartment. Dys-functional hydrogenosomes are removed by an autophagic process and further digestedin lysosomes.

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1Introduction

During the evolutionary process, eukaryotic microorganisms appeared pre-senting special cytoplasmic organelles (Fig. 1a–d). One example was the hy-drogenosome initially found in protozoa of the Trichomonadida Order, whichcontains enzymes that participate in the metabolism of pyruvate formed dur-ing glycolysis and was the site of molecular hydrogen and ATP formation(reviewed in Müller 1993). In trichomonads, hydrogenosomes have been rec-ognized by light microscopists for a long time, as paraxostylar and paracostalgranules, due to their proximity to the axostyle (bundle of microtubules) andthe costa, a periodic proteinaceous structure (Fig. 1c,d). Only biochemistryrevealed their functional significance, which showed molecular hydrogenproduction as a metabolic end product. Consequently, they were named hy-drogenosomes by Lindmark and Müller, in 1973. Hydrogenosomes present anunusual function: under anaerobic conditions they produce molecular hydro-gen by oxidizing pyruvate to malate (Müller 1993).

2Where are Hydrogenosomes Found?

The hydrogenosome is an unusual organelle found in some anaerobic fungi(Fig. 1a) (Yarlett et al. 1986; Marvin-Sikkema et al. 1992; Benchimol et al.1997), in rumen ciliates (Fig. 1b) (Yarlett et al. 1981, 1984; Snyers et al. 1982),in protozoa of the Trichomonadida Order (Fig. 1c) (Lindmark and Müller1973; Kulda et al. 1987), and also in some free-living ciliates (van Bruggenet al. 1984). The hydrogenosome-containing microorganisms do not presenttypical mitochondria (Embley et al. 2003). Until today, hydrogenosomes havenot been found in multicellular animals or plants, nor in other anaerobicprotists, such as amoebas and giardias. The most extensive studies of thisorganelle have been carried out in the trichomonad species. The distribu-tion of hydrogenosomes in different Phyla raised questions concerning theirevolutionary origin.

Fig. 1 �General view of different cells that possess hydrogenosomes (H): a the anaero-bic fungus Neocallimastix, b a rumen ciliate, and c,d Tritrichomonas foetus as seen bytransmission electron microscopy (a–c) and high voltage electron microscopy (d). In d,the plasma membrane of T. foetus was removed and the preparation was critical pointdried and observed in a high voltage electron microscope with 1000 kV of electron ac-celeration. Note that hydrogenosomes are preferentially located along the axostyle (Ax)and costa (C). ER, endoplasmic reticulum; GL, glycogen granules; F, anterior flagella;RF, recurrent flagellum; N, nucleus; P, pelta; V, vacuoles. Bars: a = 2 µm; b = 300 nm;c = 500 nm; d = 1 µm. (Fig. 1a from Benchimol et al. 1997; Fig. 1b–d, Benchimol, unpub-lished)

Structure of the Hydrogenosome 77

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3Hydrogenosome Shape

In some fungi such as Neocallimastix (Figs. 1a, 4, 5b) (Yarlett et al. 1986;Benchimol et al. 1997; van der Giesen 1997), in rumen ciliates (Fig. 1b)(Yarlett et al. 1981, 1984), in trichomonads (Figs. 1c,d, 2, 5, 8, 9a, 16) (Benchi-mol et al. 1996a), and in free-living ciliates (Finlay and Fenchel 1989) hy-drogenosomes are spherical or slightly elongated granules. Using freeze-etching, filaments are seen connecting hydrogenosomes to cytoskeletal struc-tures such as the axostyle and costa structures (Benchimol et al. 2000). Insome cells, hydrogenosomes are not spherical but are very elongated struc-tures, such as those found in Monocercomonas (Diniz and Benchimol 1998),which can reach 2.0 µm in length (Fig. 2c).

Fig. 2 Routine preparation of a hydrogenosome (H) in Trichomonas vaginalis (a), Tri-trichomonas foetus (b), and Monocercomonas sp. (c). Note that in T. foetus the hydrogeno-some is spherical, enveloped by a double membrane (arrows), and presents a singleperipheral vesicle, whereas in T. vaginalis several peripheral vesicles are seen surround-ing the organelle (arrows) and in Monocercomonas the hydrogenosome is very elongated.ER, endoplasmic reticulum; N, nucleus. Bars = 100 nm. (From Benchimol 2001; Diniz andBenchimol 1998)

4Hydrogenosome Size

Hydrogenosome sizes vary according to the species or if the cell is understress or drug treatment (Figs. 3, 13) (Benchimol 1999, 2001; Madeiro andBenchimol 2004). In trichomonads without drug treatment (Figs. 1, 2, 5a,8, 9a, 16, 18), hydrogenosomes present an average diameter of 300 nm(Figs. 2a,b, 5a), but may reach 2 µm in Monocercomonas (Diniz and Benchi-mol 1998) (Fig. 2c) or when trichomonads are subjected to drug treatment.In this last situation when drugs such as metronidazol, hydroxyurea, cy-tochalasin, and others were used, abnormal or bizarre shapes and sizes were

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Fig. 3 Abnormal hydrogenosomes. Note that the hydrogenosomes (H) are not spheri-cal as in the routine preparations. They are giant and present internal compartmentsand abnormal peripheral vesicles. Dense spots are seen in the hydrogenosomal matrix,which represent calcium deposits. When cells are submitted to stress conditions, such asincubation with fibronectin (Benchimol 2001) or other drugs (Madeiro and Benchimol2004), the hydrogenosomes present such abnormal shapes and sizes, reaching 2 µm. En-larged vesicles and internal membranes are also seen (asterisks). Bars = 150 nm. (FromBenchimol et al. 1996a)

found (Figs. 3, 13) (Benchimol 1999, 2001; Ribeiro et al. 2002; Mariante et al.2003).

5Hydrogenosome Components

5.1The Hydrogenosome Envelope

It has been shown that trichomonads (Figs. 2a,b, 5a, 7c, 15b) and fungalhydrogenosomes are enveloped by two unit membranes (Figs. 4, 5b) (Benchi-mol and De Souza, 1983; van der Giezen et al. 1997; Benchimol et al. 1997).These membranes are very thin and very closely apposed to each other(Figs. 2, 4, 5, 15b). As a general rule no space is observed between the twomembranes. Each membrane has a thickness of 6 nm and presents a certainundulation (Benchimol and De Souza, 1983; Benchimol et al. 1996a). Thepresence of CaCl2 in the fixation solution, as well as the use of reduced os-mium, is important for good visualization of the two membranes (Benchimoland De Souza 1983; Benchimol et al. 1996a) (Figs. 2, 4, 5). When the sectionsare thicker the two membranes are hardly visualized.

Conventional freeze-fracture replicas revealed the presence of the twomembranes enveloping the hydrogenosomes presenting a different numberand distribution of intramembranous particles (Fig. 7a–c) (Benchimol et al.1996a; Benchimol 2001). Four fracture faces were identified: two concave facesrepresenting the P faces of the outer and the inner membranes and two con-

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Fig. 4 Thin sections of the fungal hydrogenosome (H) from Neocallimastix frontalis. Ina there is one hydrogenosome in the process of division and the inner hydrogenosomalmembrane is in process of septum formation. Note that there is another hydrogenosomewith a double membrane, which is not in process of division. In b all the hydrogenosomesare dividing, presenting internal septa. Bars = 100 nm. (From Benchimol et al. 1997)

vex faces representing the E faces of the two membranes (Fig. 7a,c). The P andE faces of the outer membrane were frequently found. Although no quanti-tative analyses were carried out, the P face seems to have a higher particledensity than the E face. The E face of the inner membrane was observed onlyin a few cases (Benchimol et al. 1996a). Special arrangements of intramem-branous particles (rosettes) were found only when isolated hydrogenosomeswere freeze-fractured, after cell fractionation (Fig. 7b) (Benchimol 2000).

Invaginations of the hydrogenosome membrane delimiting inner compart-ments were observed when trichomonads where drug treated (Figs. 2b, 3).Some of the compartments had the same morphology and electron density asthe hydrogenosomal matrix, while others had a lower density and presentedtubular structures (Benchimol et al. 1996a).

Ciliate hydrogenosomes in Metopus and Cyclidium present internal mem-branes and look like mitochondria (Fenchel and Finlay 1995). In addition, these

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Fig. 5 Thin sections of a T. foetus (a) and a fungal (b) hydrogenosome (H) from Neo-callimastix frontalis. Both hydrogenosomes are clearly enveloped by a double membrane(arrow in (a), arrowheads in (b)). Invaginations of the hydrogenosome membrane wereoccasionally observed (arrow in (a)). The black spot in (a) is a calcium deposit in theperipheral vesicle (asterisks). CW, cell wall. Bars = 100 nm. (Fig. 5a from Benchimol,unpublished; Fig. 5b, from Benchimol et al. 1997)

hydrogenosomes are also calcium stores and display a membrane potential,which are similar to features found in mitochondria (Biagini et al. 1997).

5.2The Peripheral Vesicle

A special compartment is found at the periphery of most hydrogenosomes(Figs. 2a,b, 5a, 6b, 7b,c, 8b,c, 9, 14a,c, 15). T. foetus hydrogenosomes presentone or two peripheral vesicles (Figs. 2b, 5a), whereas T. vaginalis hydrogeno-somes exhibit several vesicles at the organelle periphery (Fig. 2a). Thus, thenumber of these compartments can be useful in taxonomic studies (Benchi-mol, unpublished). The hydrogenosome peripheral vesicle varies in size andelectron density. Morphometric analysis showed that it represents 8.6% ofthe whole organelle in T. foetus. The peripheral vesicle is surrounded alongits full extension by two closely apposed unit membranes. The inner portionof this compartment is completely distinct from the hydrogenosomal ma-trix (Figs. 8c, 9b). In some preparations it appears empty (Fig. 6b), whereasin others it presents a certain content, such as when the cells are fixedin a solution containing calcium (Figs. 2a,b, 5a, 9a, 14a,c, 15a,b) or whenthe cells are submitted to freeze-fracture and deep-etching (Fig. 8b). Thiscompartment is occupied by electron-dense reaction products after variouscytochemical detections for calcium (Figs. 2a,b, 5a), phosphatases such asacid phosphatase (Fig. 11b), Mg++-ATPase, or 5′-nucleotidase (Queiroz et al.1991).

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Fig. 6 Carbohydrates in hydrogenosomes. Monocercomonas sp. after the Thiéry tech-nique (a). The hydrogenosomal membranes are positive for carbohydrates. b T. foetuscryosection labeled with gold-conjugated WGA. Hydrogenosome (H) shows that themembrane lining the peripheral vesicle, but not other portions of the organelle, is labeled.Bars = 100 nm. (Fig. 6a from Diniz and Benchimol 1998; Fig. 6b from Benchimol et al.1996a)

Fig. 7 Freeze-fracture images of hydrogenosomes from T. foetus. a Fractured cell showinga prominent Golgi (G) with several lamellae and fenestrae, as well profiles of endoplasmicreticulum (ER) in close proximity (arrows) with hydrogenosomes (H). Bar = 100 nm.b Hydrogenosomes from an isolated fraction observed by freeze-fracture. Note the clus-ters of intramembranous particles forming rosettes (arrow). The peripheral vesicle issmooth and does not present clusters of particles or rosettes. Bar = 50 nm. (From Benchi-mol et al. 2001). c Two freeze-fractured hydrogenosomes exhibiting different fractureplanes (arrows). Bar = 100 nm. (From Benchimol et al. 1996a)

Carbohydrates were cytochemically detected in hydrogenosome mem-branes using the periodic acid–thiosemicarbazide–silver proteinate tech-nique (Fig. 6a) and gold-labeled lectins, such as WGA (Fig. 6b) (Benchimol

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Fig. 8 Different views of T. foetus hydrogenosomes (H) after field-emission scanningelectron microscopy (FESEM) (a) and freeze-etching (b,c). An isolated hydrogenosomeobtained from T. foetus observed by FESEM, where details of its surface can be seen.b A calcium deposit in the peripheral vesicle (asterisk); c shows that the peripheral vesicle(arrow) presents a smooth surface, distinct from the organelle body. Bars = 50 nm. (FromBenchimol 2000)

et al. 1996a; Benchimol and Bernardino 2002). The membrane of the pe-ripheral vesicle compartment is intensely labeled by WGA-gold, indicatingthe presence of N-acetyl glucosamine (Fig. 6b). Interestingly, the membranelining the outer portion of the hydrogenosome is not labeled (Benchimolet al. 1996). The membrane surrounding the peripheral vesicle is smoothwhen analyzed in freeze-fracture and deep-etched cells (Figs. 7c, 8c) and thus

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Fig. 9 a Hydrogenosomes isolated by Percoll–sucrose density centrifugation. The hy-drogenosomes (H) are seen as spherical organelles presenting a peripheral vesicle. Bar= 300 nm. b Hydrogenosome peripheral vesicles (P) isolated from a pure hydrogenosomefraction (a), show that they form a distinct subcompartment. Bar = 100 nm. (Courtesy ofDr. José Andrés Morgado Díaz)

rather distinct from the whole organelle, which is rough (Fig. 8a,c). Withfield-emission scanning electron microscopy (FESEM), the hydrogenosomalsurface exhibits a distinct morphology with a rough surface (Fig. 8a) (Benchi-mol 2000).

In a study by Díaz and De Souza (1997), the authors were able to purifya hydrogenosomal fraction (Fig. 9a) and then another hydrogenosomal sub-fraction containing only the peripheral vesicles (Fig. 9b), showing that it isa distinct compartment. The authors used Tritrichomonas and showed thata further treatment with proteinase K solubilized the matrix components,leaving a pure peripheral vesicle fraction (Fig. 9b). The isolated peripheralvesicles maintained their flattened morphology, suggesting that each indi-vidual vesicle has its own inherent structural framework (Fig. 9b). Afterhydrogenosomal membrane solubilization, the hydrogenosome matrix ap-peared attached to the peripheral vesicle. SDS-PAGE showed that proteins of66, 45, and 32 kDa were localized in the peripheral vesicle (Díaz and De Souza1997). Western blot analysis revealed the presence of glycoproteins, witha major one of 45 kDa in the peripheral vesicle of the hydrogenosome. Theauthors concluded that the peripheral vesicle is a distinct hydrogenosomalcompartment (Díaz and De Souza 1997).

5.3The Matrix of the Hydrogenosome

The hydrogenosome matrix is homogeneous, presenting a granular appear-ance, which is different from the cytoplasmic matrix (Figs. 1, 2, 5, 8b). Thehydrogenosome matrix was described in previous studies as homogeneouslygranular, occasionally presenting a dense amorphous or crystalline core, also

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known as nucleoid (Fig. 10a) (Honigberg and Brugerolle 1990). Observa-tions indicated that this core is not a usual structure, appearing howevereither when the protozoa are incubated in the presence of drugs or whengood fixation is not achieved (Benchimol et al. 1996). The electron-densecore is frequently seen in not-well-preserved cells, a situation in which thehydrogenosome proteins could coagulate and precipitate, leading to the for-mation of the core (Fig. 10a). In healthy or well-preserved cells (Figs. 1, 2, 5),as well as in no drug-treated cells, it is very unusual to find this electron-denseamorphous core and thus it is not considered as a hydrogenosomal structureany more (Benchimol et al. 1996a).

The hydrogenosome matrix exhibited basic proteins when cytochemistryfor ammoniacal silver and PTA (phosphotungstic acid) (Fig. 10b) were used(Benchimol et al. 1982a). The granular structure of the hydrogenosome ma-trix may be clearly visualized in replicas of quick-frozen, freeze-fractured,deep-etched, and rotary-replicated cells (Figs. 8b, 18b). When the fractureplane exposed the internal portion of the hydrogenosome matrix a largenumber of particles were seen. Most of them had a diameter of 6 nm. Some,however, were larger, with a diameter of 20 nm. These particles were not ran-domly distributed; a certain orientation in their array was noted.

Fig. 10 a T. foetus showing a dense material in the hydrogenosomes (H) matrix, namedcore or nucleoid (arrow) in early literature. The nucleoid is considered a fixation ar-tifact or is a result of a disturbance in hydrogenosome metabolism, being a sign oforganelle death (Benchimol, unpublished). Bar = 300 nm. b Cytochemistry for basicproteins (PTA), showing positive reaction in the hydrogenosomes matrix (H), in theaxostyle (Ax), costa (C), and pelta (P). Bar = 300 nm. (From Benchimol et al. 1982a)

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Fig. 11 a T. foetus after cytochemistry for calcium using potassium pyroantimonate,showing an intense labeling in the hydrogenosome matrix (arrows). N, nucleus. Bar =300 nm. (From Benchimol et al. 1982b). b Cytochemical detection of acid phosphatasepresented positive reactions not only in lysosomes (L) but also in the peripheral vesiclesof the hydrogenosomes (H). Bar = 100 nm. (From Benchimol et al. 2001)

Calcium deposits are seen as electron-dense spots in the matrix of somehydrogenosomes when cells are incubated in the presence of calcium ions inthe fixative (Fig. 3a) or processed cytochemically for the localization of Ca++

(Fig. 11a), such as with the pyroantimonate technique (Benchimol et al. 1982b).When cells are stressed with drugs, the hydrogenosomal matrix, which

normally appears finely granular and homogeneous, frequently contains in-ternal elements, such as concentric membranes, internal subcompartments,vesicles, grains, and fibrils (Fig. 3). A large, dense precipitate or nucleoid(Fig. 10a) is sometimes observed in the hydrogenosome matrix, which meansthat the organelle is dysfunctional (Benchimol et al. 1996a).

6Fungal Hydrogenosomes

The hydrogenosomes of the anaerobic fungus Neocallimastix are round orelongated structures, always enveloped by two distinct but tightly apposedmembranes (Figs. 4a,b, 5b) (van der Giezen et al. 1997; Benchimol et al. 1997).N. frontalis hydrogenosomes were studied in routine preparations for trans-mission electron microscopy, freeze-fracture, and immunocytochemistry. Inaddition, images of organelle division (Fig. 4b) were very similar to those ob-served in trichomonad protozoa (Fig. 15a). These observations suggested thathydrogenosomes are homologous organelles in unrelated species, weakeninga previous hypothesis of a polyphyletic origin (Marvin-Sikkema et al. 1992)

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and reinforcing the hypothesis that fungal and trichomonad hydrogenosomesare derived from an ancestral endosymbiont.

7Proximity with Other Cellular Structures

Glycogen particles, although distributed throughout the protozoan, are con-centrated in the region where hydrogenosomes are located (Figs. 1, 4b, 14a,16a,b).

7.1Hydrogenosomes and Endoplasmic Reticulum

Not only continuity but also chemical similarities between hydrogenosomesand the endoplasmic reticulum (ER) have been demonstrated. The ER isclosely associated, though not necessarily continuous, with mitochondria,peroxisomes (Frederick et al. 1968), and plastids. Data indicated that certainmitochondrial phospholipids were formed in ER and then transferred to themitochondrion (Jungalwala and Dawson 1970). Since the hydrogenosome hasbeen considered a modified mitochondria (Embley et al. 1997) and presentsseveral similarities to this organelle, it has been suggested that at least theperipheral vesicle of the hydrogenosomes originated from the ER.

The continuities or close associations between hydrogenosomes and therough or smooth ER are often observable (Figs. 7a,c, 12a,b, 13a,b, 17a,b).Some hydrogenosomes present membranous cisternae projecting to the cy-toplasm, conferring bizarre images of the organelle (Fig. 12a). In some cases,the hydrogenosomal outer membrane displayed attached granules, similar toribosomes, when cells were treated with cytochalasin B.

An intimate association of the hydrogenosome with the ER has been de-scribed (Benchimol et al. 1996a, Benchimol 1999). It has been proposed thatthe ER could provide new membranes for hydrogenosome growth, since this

Fig. 12 Thin sections of T. foetus showing that profiles of endoplasmic reticulum (ER) areseen in continuity (arrow) to the hydrogenosomes (H). Bars = 100 nm

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organelle enlarges before its division. The ER could participate by provid-ing the membrane lipids. Three-dimensional reconstruction of a T. foetus indivision (not shown) and in interphase demonstrated a close association ofthe membranous profiles of the ER and hydrogenosomes, in a similar way tomitochondria and peroxisomes (Franke and Kartenbeck 1971).

Also it has been suggested that membranous structures seen in close con-tact with or in the vicinity of the hydrogenosomes might be a source ofmembrane lipids for hydrogenosome growth (Benchimol et al. 1996a).

8Hydrogenosome Autophagy

T. foetus under serum deprivation, drug treatment (hydroxyurea, zinc sulfate),and also in normal conditions presents autophagy (Benchimol 1999). Appar-ently normal hydrogenosomes, and also giant, abnormal hydrogenosomes andthose presenting internal membranes, were observed in the autophagic process(Fig. 13). The first event observed was the presence of cisternae of the roughER surrounding and enclosing the altered hydrogenosome, forming an iso-lation membrane (Fig. 13a–c). The hydrogenosome is first sequestered fromthe remaining cytoplasm (Fig. 13b,c) and then degraded within lysosomes(Fig. 13d). The autophagic vacuole is limited by double (Fig. 13c) or multi-ple concentric membranes and may contain a recognizable hydrogenosome(Fig. 13b–d), probably in the preliminary steps of degradation. Lysosomes fusewith the autophagic vacuole, forming a degradative structure bounded by a sin-gle membrane (Fig. 13d) and containing the hydrogenosome in various stagesof degeneration. Hydrogenosomes have been found within lysosomes, partiallydegraded, forming hydrogenosomal remnants (Benchimol 1999).

9Hydrogenosome Division

Hydrogenosomes, as almost all other organelles, always grow by prolifera-tion of preexisting organelles. Thus, each daughter cell will receive a completeset of organelles during cell division. Morphological evidence was presentedshowing that trichomonad hydrogenosomes, like mitochondria, may divideby three distinct processes: (1) segmentation (Fig. 14a,b), (2) heart-formdivision (Fig. 14c), and (3) partition (Fig. 15a,b). In the segmentation pro-cess, the hydrogenosome grows, becoming elongated with the appearance ofa constriction in the central portion (Fig. 14a,b) (Benchimol et al. 1996b).Microfibrillar structures appear to help the furrowing process, ending witha total fission of the organelle. In the partition process, rounded hydrogeno-somes, in a bulky form, are further separated by a membranous internal

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Fig. 13 Hydrogenosomes in process of autophagy. a View of a hydrogenosome froma T. foetus cell grown in a culture medium containing 4 mM hydroxyurea for 15 h. Onegiant hydrogenosome presenting an enlarged peripheral vesicle (PV) and internal mem-branes (arrows) is seen. Profiles of the endoplasmic reticulum (ER) are seen close toand surrounding the organelle. Bar = 80 nm. b Thin section of a hydrogenosome (H)enclosed by the rough endoplasmic reticulum (ER). Bar = 50 nm. c Observation ofa double-layered autophagic vacuole (arrows), containing an intact hydrogenosome (H)after high-pressure freezing and freeze-substitution. The T. foetus cell was grown undernormal conditions. Arrows point to the double membrane of the enclosing vacuole.Bar = 200 nm. d Routine preparation of T. foetus without any drug treatment. Severalhydrogenosomes (H) are seen all around, while one of them (asterisk) is seen insidea vacuolar lysosome-like structure (V), suggesting an autophagic process. Bar = 200 nm.(From Benchimol 1999)

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Fig. 14 Views of the segmentation process (a,b) of a dividing hydrogenosome (H). a Theorganelle is elongated showing a constriction in the central region (arrows). Bar =100 nm. (From Benchimol et al. 1996b). b Freeze-etching of a T. foetus hydrogenosomeafter quick freezing of living cells. Arrows point to a linear array of particles at the sep-tum constriction. Bar = 200 nm. c T. foetus showing a hydrogenosome in the process ofheart division. Bar = 100 nm. (From Benchimol and Engelke 2003)

septum (Fig. 15a,b). The division begins by an invagination of the innerhydrogenosome membrane, forming a transversal septum, separating the or-ganelle matrix into two compartments (Benchimol et al. 1996b). A necklace ofintramembranous particles delimiting the outer hydrogenosomal membranein the region of organelle division was observed by freeze-etching (Fig. 14b).

In the hydrogenosome heart-shaped process (Benchimol and Engelke 2003),the organelle gradually presents a membrane invagination on one side, leadingto the organelle division (Fig. 14c). In this case, the hydrogenosome grows an-teriorly and the process of division starts at one of the organelle poles, which

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Fig. 15 a Thin section of two hydrogenosomes (H) dividing via the partition process.The hydrogenosome (H) becomes larger and an invagination of the inner hydrogenoso-mal membrane is observed (arrowheads), gradually dividing the hydrogenosomal matrixin two compartments. Figure b shows that initially the inner membrane separates thehydrogenosome (H) in two compartments, but they are still joined by the outer hydro-genosomal membrane (arrows). Bars = 100 nm. (From Benchimol et al. 1996b)

becomes larger than the remaining organelle. Gradually, the membrane at thispole is seen inwarding. Although the organelle division begins with an inwardfurrowing, no septum is formed. The heart-shaped process is also distinct fromsegmentation, since in this process an elongation of the organelle occurs first,giving a sausage shape and a progressive attenuation of its mid-region. In theheart-shaped process, the organelle neither forms a septum nor elongates andin this way is considered a new organellar division process.

The most common form of division observed in T. foetus was the seg-mentation process, whereas partition is the most unusual division processobserved in this protist. However, in the hydrogenosomes of the fungus Neo-callimastix the most common division process was the partition (Benchimol,unpublished observations). On the other hand, in Trichomonas the heartshape was the most frequently observed division process (Benchimol, un-published observations). It is important to point out that all three formsof division could be found during any phase of the cell cycle, and that thesegmentation and heart form could be observed in the same cell. In add-ition, the organellar division is not synchronized (Benchimol and Engelke2003). Interestingly, these three modes of division and timing were also de-scribed previously in mitochondria (Tandler and Hoppel 1973). It is notknown why and how the cell chooses which form of hydrogenosomal divisionto perform.

The mitochondrial division process has been compared with the divisionin bacteria, since the mitochondrial inner membranes are likely to be of bac-

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terial descent. In bacteria a FtsZ ring adheres to the inside of the bacterialmembrane and constricts it to mediate division. The use of FtsZ homologues(van der Bliek 2000) and dynamin has been shown to participate in mito-chondrial division (Koch et al. 2005). However, these proteins have not yetbeen studied in dividing hydrogenosomes.

In hydrogenosomes, the process of division of the inner membrane is un-known, whereas the division of the outer membrane appears to be mediatedby membranous profiles, probably of endoplasmic reticulum origin. Thesestructures could aid the hydrogenosome division or participate in anotherfunction, such as providing membranes for hydrogenosome growth.

10Hydrogenosome Behavior in the Cell Cycle

Dividing hydrogenosomes can be found in all phases of the trichomonads cellcycle, similar to mitochondria (Suzuki et al. 1994). They were even observedduring the mitotic process (Benchimol and Engelke 2003). During the inter-

Fig. 16 General aspect of T. foetus in interphase (a) and under division (b–d). Notice thatin interphase the hydrogenosomes are aligned on the costa and axostyle (a), whereasduring the division they are close to the nucleus (b–d). Bars = 300 nm. (Fig. 16a fromBenchimol, unpublished; Fig. 16b,c from Benchimol and Engelke 2003; Fig. 16d fromRibeiro et al. 2000)

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phase the hydrogenosomes in trichomonads are distributed mainly along theaxostyle and costa (Figs. 1c,d, 16a) and at mitosis onset the hydrogenosomesmigrate to and around the nucleus (Fig. 16b,c). In nondividing cells, small hy-drogenosomes (100–200 nm in diameter) were found together with normalsized organelles (about 300 nm). They were located mainly along the dupli-cated axostyle (Fig. 16b). During karyokinesis, hydrogenosomes follow theaxostyle (Fig. 16d) (Benchimol and Engelke 2003; Ribeiro et al. 2000).

11Hydrogenosomes Connection to Microtubules

Delicate bridges were observed connecting the outer hydrogenosomal mem-brane to the microtubules of the axostyle. These bridges were also seenwhen fast-freezing and freeze-etching were used and could explain the hydro-genosome alignment along the axostyle. In addition, close proximity to freemicrotubules was also seen (Fig. 17), both in thin sections (Fig. 17a) andafter quick-freezing followed by freeze-etching (Fig. 17b) (Benchimol, unpub-lished).

Fig. 17 Association of hydrogenosomes and microtubules in T. foetus. a Thin section ofhydrogenosomes (H) in close proximity with microtubules (asterisks). b Freeze-etchingafter quick-freezing and rotatory shadowing showing a hydrogenosome in close associ-ation with cytoskeletal structures, probably microtubules. Bars = 200 nm. (Benchimol,unpublished)

12Immunolabeling

Hydrogenosomes can be labeled when specific antibodies anti-hydrogeno-somal proteins are used, such as anti-malic enzyme (Fig. 18) or other proteinsfound in the hydrogenosomal matrix. Interestingly, similar labeling is also ob-

94 M. Benchimol

Fig. 18 Immuno-electron microscopy showing detection of malic enzyme in the hydro-genosomes (H) of T. vaginalis. N, nucleus; bar = 100 nm. (Benchimol, unpublished)

served when antibodies anti-AP65, an anti-adhesin protein, is used. Aldereteet al. (2001) explain this result as an example of molecular mimicry and func-tional diversity.

Acknowledgements This work was supported by CNPq (Conselho Nacional de Desenvolvi-mento Científico e Tecnológico), PRONEX (Programa de Núcleo de Excelência), FAPERJ(Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro),FENORTE (Fundação Estadual do Norte Fluminense), and AUSU (Associação Univer-sitária Santa Úrsula).

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